68Ga-Labeled [Thz14]Bombesin(7–14) Analogs: Promising GRPR-Targeting Agonist PET Tracers with Low Pancreas Uptake

With overexpression in various cancers, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer imaging and therapy. However, the high pancreas uptake of reported GRPR-targeting radioligands limits their clinical application. Our goal was to develop 68Ga-labeled agonist tracers for detecting GRPR-expressing tumors with positron emission tomography (PET), and compare them with the clinically validated agonist PET tracer, [68Ga]Ga-AMBA. Ga-TacBOMB2, TacBOMB3, and TacBOMB4, derived from [Thz14]Bombesin(7–14), were confirmed to be GRPR agonists by a calcium mobilization study, and their binding affinities (Ki(GRPR)) were determined to be 7.62 ± 0.19, 6.02 ± 0.59, and 590 ± 36.5 nM, respectively, via in vitro competition binding assays. [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA clearly visualized PC-3 tumor xenografts in a PET imaging study. [68Ga]Ga-TacBOMB2 showed comparable tumor uptake but superior tumor-to-background contrast ratios when compared to [68Ga]Ga-AMBA. Moreover, [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 showed a much lower rate of uptake in the pancreas (1.30 ± 0.14 and 2.41 ± 0.72%ID/g, respectively) than [68Ga]Ga-AMBA (62.4 ± 4.26%ID/g). In conclusion, replacing Met14 in the GRPR-targeting sequence with Thz14 retains high GRPR-binding affinity and agonist properties. With good tumor uptake and tumor-to-background uptake ratios, [68Ga]Ga-TacBOMB2 is promising for detecting GRPR-expressing tumors. The much lower pancreas uptake of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 suggests that [Thz14]Bombesin(7–14) is a promising targeting vector for the design of GRPR-targeting radiopharmaceuticals, especially for radioligand therapy application.


Introduction
The gastrin-releasing peptide receptor (GRPR) is a transmembrane G protein-coupled receptor (GPCR) that is expressed in the central nervous system, gastrointestinal tract, and pancreas [1] and regulates a variety of physiological functions, such as synaptic plasticity, hormone secretion, smooth muscle contraction, and cell proliferation [1][2][3]. Furthermore, GRPR has been shown to be overexpressed in a variety of malignancies [4][5][6][7][8][9][10] and is involved in a large array of pathophysiological conditions, such as associations with some neurochemical alterations in neurological disorders, the development of malignant neoplasms, and the proliferation of cancer cells in several cancer types [1,3,[11][12][13]. The overexpression of GRPR in malignant tissues makes it a promising target for the design of targeted radiopharmaceuticals for the diagnosis and radioligand therapy of GRPRexpressing cancer.
Gastrin-releasing peptides (GRPs) and bombesin (BBN) are two natural GRPR ligands. GRPs and BBN share the same heptapeptide sequence at the C-terminus, which has been used as the targeting vector for the design of GRPR-targeting radiopharmaceuticals for cancer diagnosis and radioligand therapy for decades [14][15][16][17][18][19][20][21]. Some of the reported GRPRtargeting radioligands have been evaluated in the clinic [15][16][17][18][19][20]. However, all clinically evaluated GRPR-targeting radioligands have shown an extremely high uptake in the pancreas [15,19,20,22]. The high pancreas uptake limits the application of these GRPRtargeting radiopharmaceuticals for detecting cancer lesions adjacent to or located in the pancreas and lowers the maximum tolerated dose for targeted radioligand therapy to minimize toxicity.
The development of GRPR-targeting radiopharmaceuticals has been focused on the use of antagonist sequences as the targeting vector in the past decade, partly due to their higher in vivo stability [29], potentially higher tumor uptake due to more binding sites than those available for agonists [30], and/or less short term adverse effects [31,32]. However, agonists can be internalized upon binding to GRPR and potentially lead to longer tumor retention [1,32,33], which might be preferable, especially for use in the development of radiotherapeutic agents. We hypothesized that (1) replacing the reduced peptide bond (Leu 13 ψThz 14 ) in our previously reported Ga-TacsBOMB2, Ga-TacsBOMB3, and Ga-TacsBOMB4 ( Figure 1A) with an amide bond would restore their GRPR agonist characterizations, and (2) the resulting 68 Ga-labeled [Thz 14 ]Bombesin (7)(8)(9)(10)(11)(12)(13)(14) derivatives might retain the low pancreas uptake characteristics observed from their Leu 13 ψThz 14 analogs.
Thus, in this study, we synthesized [Thz 14 ]Bombesin(7-14)-derived TacBOMB2, TacBOMB3, and TacBOMB4 ( Figure 1C, by replacing the reduced peptide bond (CH 2 -N) between residues 13-14 (Leu 13 ψThz 14 ) with a normal amide bond. Their agonist properties were determined using an in vitro fluorescence-based calcium release assay. Their potential for imaging GRPR expression was evaluated through in vitro competition binding, positron emission tomography (PET) imaging, and ex vivo biodistribution studies in a preclinical prostate cancer model, and compared with a clinically validated GRPR agonist tracer, [ 68 Ga]Ga-AMBA ( Figure 1D).

Chemistry and Radiochemistry
DOTA-conjugated TacBOMB2, TacBOMB3, and TacBOMB4 were obtained in 30-55% yields, and their nonradioactive Ga-complexed standards were obtained in 58-82% yields. The HPLC conditions for their purification and MS characterizations are provided

Chemistry and Radiochemistry
DOTA-conjugated TacBOMB2, TacBOMB3, and TacBOMB4 were obtained in 30-55% yields, and their nonradioactive Ga-complexed standards were obtained in 58-82% yields. The HPLC conditions for their purification and MS characterizations are provided in Tables S1 and S2. Gallium-68 labeling was conducted in HEPES buffer (2 M, pH 5.0). After HPLC purification, 68 Ga-labeled TacBOMB2, TacBOMB3, and AMBA were obtained in 51-80% decay-corrected radiochemical yields with 234-322 GBq/µmol molar activity and >95% radiochemical purity. The HPLC conditions for their purification and quality control are provided in Table S3.
We further determined the binding affinities of these three GRPR-targeting ligands by conducting an in vitro competition binding assay. The K i values of Ga-TacBOMB2 (7.62 ± 0.19 nM) and Ga-TacBOMB3 (6.02 ± 0.59 nM) were comparable, while Ga-TacBOMB4 showed a much poorer binding affinity to GRPR (K i = 590 ± 36.5 nM). This finding is consistent with our previous report that replacing D-Phe in Ga-TacsBOMB2 with D-2-Nal doesn't affect the binding affinity towards GRPR, while replacing D-Phe with D-Tpi leads to a significantly lower binding to GRPR. One possible explanation is that the free rotation of the Pip linker and the Ga-DOTA complex is compromised by the rigidity of the secondary amino group of D-Tpi, which results in a significant loss of binding affinity to GRPR.
In vivo stability studies revealed that [ 68 Ga]Ga-AMBA was more stable than [ 68 Ga]Ga-TacBOMB2 and [ 68 Ga]Ga-TacBOMB3 in mouse plasma, as their intact fractions were 39.4 ± 10.8, 12.7 ± 2.93 and 27.3 ± 4.84%, respectively, at 15 min post-injection ( Figures S1-S3). This indicates that the slightly higher tumor uptake of [ 68 Ga]Ga-AMBA may also owe to its better in vivo stability other than its better binding affinity toward GRPR. When comparing [ 68 Ga]Ga-TacBOMB2 with our previously reported [ 68 Ga]Ga-TacsBOMB2, [ 68 Ga]Ga-TacsBOMB2 was much more stable in vivo, with 83.3 ± 1.15% remaining intact at 15 min post-injection. This suggests that replacing the reduced peptide bond (Leu 13 ψThz 14 ) with an amide bond results in potential cleavage site(s) for endogenous peptidases, leading to a reduction in stability. However, this also emphasizes that there is potential for improvement for [ 68 Ga]Ga-TacBOMB2 and [ 68 Ga]Ga-TacBOMB3 if their in vivo stability can be enhanced, likely by substituting some of the amino acids in the targeting sequences with their unnatural amino acids analogs.

General Methods
AMBA and Ga-AMBA were synthesized following published procedures [36,37]. All the other chemicals and solvents were purchased from commercial sources and used without further purification. GRPR-targeting peptides were constructed on solid phase using an AAPPTec (Louisville, KY, USA) Endeavor 90 peptide synthesizer. The purification and quality control procedures for DOTA-conjugated peptides and their nat Ga/ 68 Ga-complexed analogs were performed on Agilent (Santa Clara, CA, USA) HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (220 nm), and a Bioscan (Washington, DC, USA) NaI scintillation detector. The operation of Agilent HPLC systems was controlled using the Agilent ChemStation software version C.01.05. A semipreparative column (Luna C18, 5 µm, 250 × 10 mm) and an analytical column (Luna C18, 5 µm, 250 × 4.6 mm), purchased from Phenomenex (Torrance, CA, USA), were used for purification and quality control. The HPLC eluates were collected and lyophilized with a Labconco (Kansas City, MO, USA) FreeZone 4.5 Plus freeze-drier. The MS analyses of DOTA-conjugated peptides and their nat Ga-complexed analogs were conducted with a Waters (Milford, MA, USA) Acquity QDa mass spectrometer equipped with a 2489 UV/Vis detector and an e2695 Separations module. C18 Sep-Pak cartridges (1 cm 3 , 50 mg) were purchased from Waters (Milford, MA, USA). 68 Ga was eluted from an ITM Medical Isotopes GmbH (Munich, Germany) generator, and purified according to the previously published procedures using a DGA resin column from Eichrom Technologies LLC (Lisle, IL, USA) [38]. The radioactivity of the 68 Ga-labeled peptides was measured using a Capintec (Ramsey, NJ, USA) CRC ® -25R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies was counted using a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.

Synthesis of Nonradioactive Ga-Complexed Standards
The nonradioactive Ga-complexed standards were synthesized by incubating DOTAconjugated precursor (1 eq.) and GaCl 3 (5 eq.) in NaOAc buffer (0.1 M, 500 µL, pH 4. 2-4.5) at 80 • C for 15 min. The reaction mixture was then purified via HPLC (semi-preparative column, flow rate: 4.5 mL/min). The HPLC eluates containing the desired peptide were collected and lyophilized. The HPLC conditions, retention times, isolated yields, and MS confirmations of the nonradioactive Ga-complexed standards are provided in Table S2.

Synthesis of 68 Ga-Labeled Compounds
The radiolabeling experiments were performed following previously published procedures [38][39][40]. Purified 68 GaCl 3 in 0.5 mL of water was added to a 4 mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and 10 µL of precursor solution (1 mM). The radiolabeling reaction was carried out under microwave heating for 1 min before being purified by HPLC using the semi-preparative column. The eluate fraction containing the radiolabeled product was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge that was pre-washed with ethanol (10 mL) and water (10 mL). After washing the C18 Sep-Pak cartridge with water (10 mL), the 68 Ga-labeled product was eluted off the cartridge with ethanol (0.4 mL) containing 1% ascorbic acid and diluted with PBS containing 1% ascorbic acid for imaging and biodistribution studies. Quality control was performed using the analytical column. The HPLC conditions and retention times are provided in Table S3. The tracers were obtained in 51-80% decay-corrected radiochemical yields with 234 to 322 GBq/µmol molar activity and >95% radiochemical purity.

LogD 7.4 Measurement
LogD 7.4 values of [ 68 Ga]Ga-TacBOMB2, [ 68 Ga]Ga-TacBOMB3, and [ 68 Ga]Ga-AMBA were measured using the shake flask method, as previously reported [38]. Briefly, aliquots (2 µL) of the 68 Ga-labeled peptides were added into a 15 mL falcon tube containing 3 mL of n-octanol and 3 mL of 0.1 mol/L DPBS (pH 7.4). The mixture was vortexed for 1 min and then centrifuged at 3000 rpm for 15 min. Samples of the n-octanol (1 mL) and buffer (1 mL) layers were collected and measured in a gamma counter. LogD 7.4 was calculated using the following equation: LogD 7.4 = log 10 [(counts in the n-octanol phase)/(counts in the buffer phase)].

Cell Culture
The PC-3 cells obtained from ATCC (via Cedarlane, Burlington, Canada) were cultured in RPMI 1640 medium (Life Technologies Corporations) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . The cells were confirmed pathogen-free via an IMPACT Rodent Pathogen Test (IDEXX BioAnalytics). Cells grown to 80-90% confluence were washed with sterile DPBS (pH 7.4) and collected after 1 min of trypsinization. The cell concentration was counted in duplicate using a hemocytometer and a manual laboratory counter.

In Vitro Competition Binding Assay
PC-3 cells were seeded in 24-well, poly-D-lysine plates at 2 × 10 5 cells/well 24-48 h prior to the assay. The growth medium was replaced with 400 µL of reaction medium (RPMI 1640 containing 2 mg/mL of BSA, and 20 mM of HEPES). Then, the plates were incubated for about 60 min at 37 • C. Ga-complexed nonradioactive standards of TacBOMB2, TacBOMB3, TacBOMB4, and AMBA in 50 µL of reaction medium with decreasing concentrations (10 µM to 1 pM) and 50 µL of 0.011 nM [ 125 I-Tyr 4 ]Bombesin were added into the wells, followed by incubation with moderate agitation for 1 h at 36 • C. Cells were gently washed with ice-cold PBS twice, harvested by trypsinization, and counted for radioactivity on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter. Data were analyzed using nonlinear regression (one binding site model for competition assay) with GraphPad (San Diego, CA, USA) Prism 8.4.3 software.

Ex Vivo Biodistribution, PET/CT Imaging and In Vivo Stability Studies
PET/CT imaging, biodistribution, and in vivo stability studies were conducted on male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG) mice, following previously published procedures [38,[41][42][43]. The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia (protocol number A20-0113, approved on 30 September 2020). The mice were anesthetized by inhalation of 2.5% isoflurane in 2 mL/min of oxygen and implanted subcutaneously with 5 × 10 6 PC-3 cells (100 µL; 1:1 PBS/Matrigel) behind the left shoulder. Mice were used for PET/CT imaging and biodistribution studies when the tumor grew to 5-8 mm in diameter over around 4 weeks.
PET/CT imaging experiments were performed on a Siemens (Knoxville, TN) Inveon micro PET/CT scanner. The tumor-bearing mouse was injected with 3-5 MBq of 68 Galabeled tracer through a lateral caudal tail vein under anesthesia, followed by recovery and roaming freely in its cage during the uptake period. At 50 min post-injection, a 10 min CT scan was conducted first for the localization and attenuation correction after segmentation for reconstructing the PET images, followed by a 10 min static PET imaging acquisition.
For in vivo stability studies, 7-9 MBq of [ 68 Ga]Ga-TacBOMB2, [ 68 Ga]Ga-TacBOMB3, or [ 68 Ga]Ga -AMBA was injected via the lateral caudal vein into healthy male NRG mice (n = 3). At 15 min post-injection, mice were sedated and euthanized, and urine and blood were collected. The plasma was extracted from whole blood by adding CH 3 CN (500 µL), vortex, centrifugation, and the separation of supernatant. The plasma and urine samples were analyzed via radio-HPLC using the conditions for quality control of these 68 Ga-labeled radioligands (Table S3).

Conclusions
Replacing the (Leu 13  is a promising vector for the design of GRPR-targeting radiopharmaceuticals, particularly for radioligand therapy applications to minimize toxicity to the pancreas.

Patents
The compounds disclosed in this report are covered by a recent US provisional patent application (Serial number 63/323,831; filing date: 25 March 2022). Lei Wang, Zhengxing Zhang, Ivica Jerolim Bratanovic, François Bénard, and Kuo-Shyan Lin are listed as inventors of this filed patent.